[Methods in Molecular Biology™] Methods in Membrane Lipids Volume 400 || Langmuir Films to...

29

Langmuir Films to Determine Lateral Surface Pressure on Lipid Segregation

Antonio Cruz and Jesús Pérez-Gil

SummaryInterfacial monolayers used as membrane models have become a practical technique to obtain detailed informa-

tion about lateral processes taking place in the membrane. These monolayers are particularly useful to study theinteractions and parameters governing lateral distribution of lipid and protein species and the association of differ-ent molecules with membrane surfaces. In the last few years, these classical models have been complemented by awhole collection of new techniques that are able to provide spatial information on the structure of the interfacialphospholipid-based films at both microscopic and nanoscopic scales. In the present chapter, some detailed proto-cols are described on how to prepare phospholipid Langmuir films, obtain structural information from their com-pression isotherms, and study their structure either in situ at the interface or on transfer onto solid supports byapplying different microscopy techniques. The use of exogenous fluorescent probes and the extraction of qualita-tive and quantitative information from epifluorescence microscopy images are particularly addressed.

Key Words: Epifluorescence microscopy; interfacial monolayer; Langmuir films; Langmuir–Blodgettfilms; lipid domains; liquid-condensed; liquid-expanded; liquid-ordered; phase diagram; phase segregation;phase transition; rafts; surface tension.

1. IntroductionLateral segregation of lipids in biological membranes is being recently proposed as a gen-

eral mechanism governing different cellular processes, such as signal transduction and inter-and intracellular trafficking (for a comprehensive review, see refs. 1–3). The development oftechniques to evaluate lateral membrane structure has been highly demanded in this respect.Lipid monolayers were already at the basis of the historic observation by Benjamin Franklinthat the waves in a pond could be reduced by spreading olive oil at the surface, but the pio-neer work by Irving Langmuir during the first years of the 20th century was the one that setthe fundamentals of modern surface balances, monolayer preparation, and the basis for thethermodynamic analysis of the behavior of such films. Langmuir films have been extensivelyused as membrane models to study lipid lateral organization and lipid–protein interactions,providing some advantages when compared with liposomes.

Interfacial monolayers allow a precise control of some factors affecting lipid structure,such as accurate composition, lateral pressure, and packing state. The precise spatiallocalization of monolayers at the air–water interface allows application of differenttechniques particularly suited to visualize Langmuir films at different scales, permitting theidentification of lateral segregation on membrane-based lipid mixtures and the recognitionof lipid–protein interaction sites. Transference of Langmuir films onto solid supports facilitates

From: Methods in Molecular Biology, vol. 400: Methods in Membrane LipidsEdited by: A. M. Dopico © Humana Press Inc., Totowa, NJ

439

the application of other complementary techniques to the analysis of the structure of mono-layers as membrane models.

2. MaterialsThe best available quality materials must be used to ensure good results when preparing

Langmuir films. Water for the preparation of buffers must be, at least, double distilled. Deionizedwater (Milli-Q quality, Millipore, Billerica, MA) could be used, but a further distillation step per-formed in the presence of potassium permanganate to oxidize organic molecules is highly rec-ommended. It is important to use freshly prepared water; nevertheless, water could be preservedat 4°C for a few days. Plastic recipients must be avoided to prepare or store materials.

1. Pure water is recommended as the aqueous subphase. When control of pH and pI is necessary,the experiment could be carried out using buffers made with nonsurface-active compounds. It isnecessary to take into account that the presence of ions usually yields interactions at the filmsaffecting the slope of the isotherms. Tris-HCl buffer at low concentrations (~5 mM) has beenused to work at pH near neutrality. When preparing transferred films, the presence of salts maylead to crystal formation during the dried out period.

2. Solutions of the different film components must be prepared using high-performance-liquidchromatography grade solvents. Chloroform (Chl) and methanol (MetOH) mixtures are fre-quently preferred to prepare phospholipid-based solutions; Chl/MetOH 3:1 (v/v) is a broad sol-vent optimal for most phospholipids.

3. Lipids: analysis by thin-layer chromatography could be required to check for purity.Dipalmitoylphosphatidylcholine (DPPC), whose films show a well-defined Π-area isotherm, isa good standard phospholipid that could be used as training lipid or to check periodically thatthe system is clean enough and ready to work.

4. Fluorescent dyes: 7-nitrobenz-2-oxa-1,3-diazole-phosphatidylcholine (NBD-PC), rhodamine-PE,DiIC18, Bodipy-PC (Molecular Probes, Junction City, OR), and so on. Dyes added as traces to thelipid solutions must be kept at proportions not higher than 0.5–1% M with respect to phospholipids(and preferably in the lowest range), to prevent spurious effects on the monolayer structure.

5. Paper or platinum flags may be used to measure surface tension. To get optimal contact angle,platinum flags must be scratched with sandpaper before taking them to the flame for cleaning.When using papers flags, they should be frequently replaced to avoid contamination betweendifferent experiments.

6. Glass cover slips: cut at the proper size (20 × 60 mm2), are good substrates to transfer films forfluorescence applications. Molecularly flat substrates such as freshly exfoliated mica sheets orsilicon wafers should be used for atomic force microscopy (AFM).

3. Methods3.1. Langmuir Films

3.1.1. Layer Material and Solvent Selection

Any amphipathic material can be used to prepare an interfacial monolayer. This includeslipids, detergents, polypeptides, polymers, and so on. An amphipathic molecule contains ahydrophilic region, which orientates toward the water subfase, and a hydrophobic regionexposed to the air side once at the water–air interface. Amphipathic compounds are able toform single-molecule thick layers at the interface. Molecules forming Langmuir films mustbe insoluble in water. Although water-soluble amphiphiles such as detergents could also forminterfacial monolayers, these films sustaining a dynamic equilibrium with the bulk subphase.

The structure and properties of these solubilizable monolayers (termed Gibbs monolayersto differentiate them from true Langmuir films), are in tight dependence with the subphase

440 Cruz and Pérez-Gil

composition and the bulk↔surface equilibrium conditions. On the other hand, Langmuirmonolayers are characterized by the property of their molecules being strictly confined at theair–liquid interface. This feature is the first determinant to select the kind of solvents to useto manipulate monolayer components. To allow for interfacial lipid deposition without lossof material toward the subphase, the solvent should be also immiscible with water. Finally,the carrying solvent has to be eliminated from the surface by evaporation; thus, very volatilesolvents are preferred. Chl/MetOH 3:1 (v/v) mixture, for instance, is a good solvent for dissolv-ing phospholipid-based mixtures to prepare the kind of films frequently used as models ofbiological membranes.

Unless explicitly stated, most of the procedures described in this chapter make referenceto the preparation and use of phospholipid interfacial phospholipid films. Preparation of layersof other amphiphiles different than phospholipids may require other solvent vehicles, such asethyl-ether, benzene, or n-hexane. In some cases, when the influence of the hydrophilic partof the molecule makes it insoluble in nonpolar solvents, the proportion of alcohols like MetOHor ethanol could be increased. In these cases, care must be taken to avoid loss of material intothe subphase. Proteins or peptides can be deposited into the interface through dissolution inpolar solvents such as dimethyl-sulphoxide or acetone. However, the high miscibility of thesesolvents with water makes it difficult, if not impossible, to avoid a partial loss of the proteininto the bulk phase.

Isopropanol-based solvent mixtures may be a good alternative (4). It is important to takeinto account that the structure of proteins and peptides may be irreversibly affected by theexposure to some of these solvents, producing not easily explainable results.

To prepare the films, phospholipid organic solutions at concentrations between 0.1 and2 mg/mL can be used. The concentration must be selected taking into account the amount ofmaterial to be initially applied, which is also related to the initial surface area of the trough atmaximal opening of the balance. Diluted lipid solutions may lead to the application of largevolumes of carrying solvent onto the surface, needing long times to complete solvent evapo-ration and increasing the risk of accumulation of solvent trace contamination. Concentratedlipid solutions, more than 2 mg/mL, could make it difficult to accurately manipulate the volumeto be applied, increasing the experimental errors on isotherm calculations. Moreover, too concen-trated solutions could yield an undesired increase in the solution density, potentially causingloss of material sunken toward the subphase during monolayer deposition.

3.1.2. Surface Balance

3.1.2.1. TROUGH TYPES

In general, troughs are typically made from polytetrafluoethylene (PTFE). This is a hydrophobicand highly inert material, allowing easy cleaning of the trough with organic solvents orinorganic acids. Some surface balances are differentiated by the mechanism set to compressand expand the film at the interface. The most common and simplest system is a PTFE bar,which is used as a single movable barrier that scans the subphase meniscus while maintain-ing the Langmuir film confined in the surface (see Note 1). This mechanism is good enoughfor most applications, but the subphase meniscus in this device acts as the monolayer con-tainer. Thus, special care is required to maintain the surface level constant against subphaseevaporation (Note 2). The use of balances equipped with bar meniscus barriers is usuallyenough to study monolayers in the pressure ranges thought to mimic lipid packing in free-standing membranes (in the order of ~30 mN/m). However, this type of barriers does not

Lipid Segregation in Langmuir Films 441

overcome some problems when the films are taken to high enough pressures (>40 mN/m),because surface tension may not be enough to sustain the meniscus, and leakage of thesurface material through the barrier and through the edges of the trough may occur.

To avoid these problems, other compression mechanisms have been implemented. The solutionof dipping the barrier inside the trough walls could be enough to avoid some inconveniences,but some leakage through the contact edges of barrier and trough may still take place if thecontact zone is not tight enough. The best solution for applications requiring full access to thehighest surface pressure regimes is the use of balances with a continuous perimeter barrier.In these balances, the whole film is confined inside an edge-free, continuous PTFE ribbonbelt or an articulated single PTFE piece, which is deeply immersed into the subphase. Thus,the change of available surface during compression/expansion is achieved by modifying theshape of the area engulfed by the flexible barrier.

3.1.2.2. TROUGH CLEANING

The trough may be cleaned in four steps:

1. Dismount the balance; immerse the barriers and any mobile pieces into enough volume ofChl/MetOH 3:1 solvent solution.

2. Fill the trough with enough volume of Chl/MetOH 3:1 and wait for 5 min.3. Remove the solution and fill the trough with MetOH to remove Chl traces.4. Wash three times with ddH2O.

Addition of 2% of 0.1 N HCl to the Chl/MetOH 3:1 cleaning solution could aid to eliminateprotein remains. Wiping the trough with a clean paper impregnated with Chl/MetOH beforeproceeding to solvent washing could be required to remove material adhered to the trough.

3.1.3. Interfacial Deposition

3.1.3.1. FILMS PREPARED FROM ORGANIC SOLVENT SOLUTIONS

Surface pressure measurements and compression parameters in modern troughs are undercontrol of a computer and proper software. Before starting acquisition of the isotherm, filmscan be formed by spreading the amphiphiles in solvent solutions according to the followingprotocol:

1. Fill the trough with clean subphase and wait until the chosen temperature of the subphase hasbeen achieved.

2. Clean the flag. Platinum flags can be easily cleaned by flaming. Paper flags should be exhaus-tively washed with the subphase solution before wetting.

3. Calibrate the force transducer of the balance using known weights and adjust the program tomonitor surface pressure (Π) (see Note 3). Determination of surface pressure requires calcula-tion of the contact edge between flag and water. Flag weight is discounted at the initial step.

4. Hang the flag from the force transducer and adjust the balance to measure 0 mN/m surface pressure.Dip the flag 1 mm into the subphase and measure the apparent surface pressure (see Note 4).Because air is used as reference, the measurement in terms of surface pressure must read nowapproximately the negative value of the surface tension of the subphase (~72 mN/m in cleanwater), with some deviation from the theoretical value because of the effect of the floating forceproduced by the immersed part of the flag. For higher accuracy of this control measurement, theflag must touch the surface without any immersed volume; it is not so critical to obtain the pres-sure isotherm because floating force will be initially discounted. If the apparent baseline surfacepressure value is far from the expected, check the subphase purity or reclean the system.


5. Set the balance to 0 mN/m and close the barrier to compress the interface. If the interface is reallyclean, no changes in surface pressure on compression should be observed. A limited increase inpressure is usually observed as a result of the presence of some trace of surface active moleculesat the minimum area position; clean then the interface by sucking the surface with a pipetconnected to a vacuum pump. Repeat this procedure as many times as necessary until no changesin surface pressure are observed on compression.

6. After those cleaning procedures, a lack of increase in surface pressure indicates that the air–liquidinterface is free of any surface active molecule. If a systematic repetition of step 5 does not endwith good base lines, contamination of the subphase with some amphipathic material that iscontinuously adsorbing into the interface during the cleaning steps most likely occurred. In thiscase, check the trough cleaning, wash it again with double distilled water, and change thesubphase with clean freshly prepared solution.

7. Once the surface is thoroughly clean, take the appropriate amount of the phospholipid organicsolution with a microsyringe; deposit the solution drop-by-drop by placing the microsyringe tipend at a few millimetres from the water surface. Deposit lipid at the interface until the surfacepressure increases from 0 to 0.1–0.2 mN/m, measuring accurately the volume that is finallyapplied. The volume measured may be used as a reference for successive experiments, and hasto be strictly known to obtain accurate Π-area (Π-A) isotherms. An estimation of the amount oflipid required to start obtaining the isotherm can be derived from reference isotherms of thelipid. When a relatively large volume of lipid solution has to be spread, deposition of drops ondifferent positions over the surface is recommended, waiting some time to allow for solventevaporation between drop depositions.

8. Once all the solvent volume has been spread, wait 10 min to allow for solvent evaporation and lipidextension. However, longer times might be required, depending on the solvent and volume applied.

3.1.3.2. FILMS PREPARED FROM AQUEOUS SUSPENSIONS

It may be useful to obtain interfacial phospholipid-based films that also contain proteins.Interaction of the proteins with lipid vesicles in a previous step permits forming films fromthese lipid–protein suspensions. Proteins could have been isolated or reconstituted in lipidsfor a better stabilization of their native structure. Preparation of films by spreading aqueouslipid/protein suspensions assumes that at least part of the lipid and protein molecules are trans-ferred from the bilayered structures into the interface (5). Such transfer is more efficient insome system than others. Some other considerations must be taken into account:

1. Vesicle suspensions are prepared in water; special care has to be taken during lipid deposition ofthese aqueous suspensions. Apply the material by forming a small drop on the syringe tip andlet the drop touch the surface to allow for vesicle spreading. Because loss of some material intothe subphase is unavoidable, more material is needed when using suspensions than when usingorganic solvent solutions (6).

2. The subphase volume is much higher than the volume applied; thus, in practical terms, the mate-rial lost in the subphase during the deposition procedure does not further reach the interface. Theamount of material that is finally transferred onto the surface then depends on the initial adsorp-tion efficiency (7). This means that only suspensions that adsorb quickly enough at the interfaceare useful to prepare lipid/proteins films. For example, vesicles made only with lipids bearinglong hydrocarbon tails, such as DPPC, are very stable and do not practically adsorb into theinterface. The morphology of the lipid vesicles used can also be important: small unilamellarvesicles prepared by strong sonication are intrinsically unstable and adsorb more efficiently intothe interface than large unilamellar vesicles.

3. Partial loss of material because of incomplete transfer must be taken into account when comparingdifferent isotherms, because the actual material on the surface cannot be easily calculated. Anapparent area per lipid molecule could be estimated from the volume applied, but isotherms


calculated this way can only be compared when obtained from materials having comparableinterfacial adsorption efficiencies. Interfacial adsorption kinetics may need to be analyzed inorder to explain differences on area per lipid molecule data (7).

3.1.4. Π-A Isotherms

The structure of an interfacial monolayer formed by a given lipid mixture depends basi-cally on the temperature, the surface concentration of lipid molecules, and the lateral pres-sure of the film. Surface pressure vs area (Π-A) isotherms are usually recorded by changingthe area occupied by the monolayer at the air-liquid interface, while the surface pressure iscontinuously monitored. Assuming that all applied lipid has been transferred and is confinedto the air–liquid interface, the area per lipid molecule can be easily calculated from theknown total area of the trough, the sample concentration, and the initial volume of spread-ing solution that was applied. This assumption is reasonable if the film has been formedcarefully as described in Subheading 3.1.3.1., but it can be difficult to confirm when thefilms are formed from aqueous vesicle suspensions or from lipid solutions containing highproportions of polar solvents.

All modern Langmuir troughs have motorized barriers controlled by software, allowing theselection of start and end positions, as well as the barrier speed. To obtain an isotherm, fill thetrough with clean bulk solution and wait until the required temperature is attained. Clean theinterface, open the barrier, and spread the lipid as described in Subheading 3.1.3.1. Select aproper compression rate and start the compression program (see Note 5). An incomplete equi-libration of the film that may occur during relatively fast compression may affect the slope ofthe isotherms, depending on the barrier speed (Note 6). On the other hand, the apparent pat-tern of the compression-driven structural transitions occurring in the films is largely affectedby the compression rate. Rapid compression usually produces higher number of condensedlipid domains smaller than those seen produced in slowly compressed isotherms (8). Thus, thecompression speed used to take the films to the desired pressures must be taken especially intoconsideration when comparing epifluorescence images.

A typical Π-A isotherm of a DPPC film is shown in Fig. 1. During compression, surface pres-sure increases as a function of area reduction and associated increase in lipid concentration at theinterface. The different slopes in the segments of the curve indicate different compressibilitydegrees of the film at the different pressure regimes. Abrupt changes in the curve are commonlyassociated with compression-driven structural transitions occurring in the lipid films. Typically,four different two-dimensional (2D) phases have been described in isotherms from single lipidcomponents: gas (G), liquid-expanded (LE), liquid-condensed (LC), and solid phase (S). At lowpressures, the lipid behaves as a 2D G phase, characterized by very low density of disorderedlipid molecules at the interface. As compression is increased, the available free surface is reduceduntil the molecules are forced to touch each other. At this point, surface pressure starts to riseand the lipid adopts a LE phase (see Note 7). Structurally, this phase shows up because of areorganization of the molecules that form the monolayer, i.e., with the phospholipid head groupsfacing the aqueous bulk phase and the phospholipid hydrocarbon tails oriented almost perpen-dicularly to the interface plane (yet with some intrinsic flexibility because of trans–gauche iso-merizations of the hydrocarbon chains). At higher pressures, the lipid may get into a LC phase,with a more compact ordering (see Note 8). The steeper slope of this segment of the isothermsreveals that this phase possesses low compressibility. In the LC phase, the lipid molecules aretightly packed, exhibiting highly limited molecular movements in the plane of the monolayer.


Coexistence of regions having LE and LC phases in the films is usually associated with amarked plateau in the isotherm, where compression produces very slight increases on surfacepressure. Along this plateau, which behaves as a real phase transition edge of the phase dia-gram, the work of compression is used to promote the change from LE to LC phase.Morphologically (see Fig. 3) the coexistence of the two phases along this plateau is observedas the formation of lipid domains in LC phase surrounded by a background of LE phase. Thisconstant pressure plateau is characteristic of a 2D phase transition and depends on tempera-ture. Higher temperatures shift the transition pressure to higher values.

If surface pressure is increased further, a solid phase can be reached, observed as a kink inthe isotherm to a segment with increased slope and practically no compressibility. There issome controversy about the significance of this phase because the translational movement ofthe molecules at the interface is similar to that observed in the LC phase. X-ray diffractionshows that the main difference between the two states can affect the tilt of the hydrocarbonchains of the phospholipid molecules (9). Compression of the solid phase beyond a certainlimit produces the collapse of the film with loss of material from the monolayer and forma-tion of three-dimensional (3D) structures beneath or above the interface. At this point ofthe isotherm, surface pressure is no more affected by compression. This pressure is known asthe collapse pressure of the film. A monolayer made of pure DPPC, for instance, is able toreduce surface tension to near 0 mN/m (reaching above 70 mN/m surface pressure) beforecollapsing. The area occupied per lipid molecule at LC or LE states can be obtained byextrapolating the curve segment corresponding to any of those phases to Π = 0 mN/m.


Fig. 1. Surface pressure–area (Π–Α) compression isotherms, and the 2D phases associated, ofsome illustrative interfacial phospholipid films. Films made from a single saturated phospholipidspecies (DPPC) (left panel) transit at low pressures from a gas (G) to a LE phase. When compressedat temperatures lower than 40°C, a conspicuous plateau in the isotherm is the result of the LE/LCtransition ending first in a pure LC phase, and then in a 2D solid-like (S) film, collapsing (C) at pres-sures higher than 70 mN/m. Films made from an unsaturated phospholipid species, such as egg yolkPC (central panel), collapse directly from the LE phase. Isotherms obtained from films made up ofa full lipid mixture such as, for instance, the organic-extracted fraction of pulmonary surfactant (rightpanel) are much more complex to interpret, with contribution of LE and LC phases but also ofliquid-ordered-like (LO) regions. Beyond certain pressures, these complex isotherms may includesqueeze-out plateaus originated by the partial exclusion of some of the components or the whole filmaway from the interface.

Isotherms from monolayers made of a mixture of lipids are more complex to interpret thanthose from single lipid films. Theoretically, the isotherm obtained from a mixture of two ormore lipid components should be comparable with the algebraic sum of the isotherms offilms made of the single components normalized according to their relative molar fraction.Any deviation of this theoretical curve is indicative of the existence of attractive or repulsiveforces between the different molecular components. Typical curves could be obtained plot-ting the area occupied by the lipid mixture at a given pressure vs the molar fraction of one ofthe components. Ideally, a straight line must be found when plotting the area per lipid mole-cule of any of the components against its molar fraction. A negative deviation from this the-oretical line could indicate a potential interaction between the different components (10).

In mixed films, mutual interaction or segregation of the different components can yield struc-tures or phases not existing in single lipid monolayers. Lateral immiscibility or segregation maynot be easily detected in the isotherms of these mixed films in the form of plateaus, and some-times it can only be observed when structure is analyzed. On the other hand, plateaus showingup in mixed systems could be owing to 2D to 3D structural transitions, including exclusionof part of the components of the layer during lateral compression. Methods that allow formicroscopic observation of the monolayer usually help to interpret the data obtained fromthe isotherms.

3.2. Langmuir–Blodgett Films

Katharine Blodgett was the first to study the transfer of monolayers from the liquid surfaceonto a solid substrate crossing the liquid interface vertically. Blodgett’s work, carried out underLangmuir supervision, included characterization of parameters that control monolayer transferand some physical properties of the transferred films. Some later studies demonstrated thatLangmuir–Blodgett (LB) films transferred at constant surface pressure may conserve most ofthe structural information of the Langmuir monolayers as they are at the interface.

3.2.1. Selection and Cleaning of Solid Supports

The LB procedure allows transferring the interfacial films either from the hydrophobic orthe hydrophilic side of the monolayer by selecting the support material and the direction ofthe transfer. Hydrophilic substrates immersed into the subphase and raised through the mono-layer plane adsorb the lipid monolayer, producing a film in which the lipid head groups interactwith the substrate and the hydrocarbon chains are exposed to air (see Fig. 2). Films trans-ferred with the opposite orientation may be obtained by using a hydrophobic material as sub-strate and reversing the transfer direction. The hydrophilic transfer is preferred because ofinherent problems associated with transfer to hydrophobic supports. The adherence of lipidacyl chains to hydrophobic substrates is intrinsically weak, and as a consequence, the transferof lipid films to those supports is efficient only from relatively dense monolayers compressedabove certain pressures. Depending on the desired application, a variety of supports can beused, including polymers, metal, or silicon wafers. Inert substrates as glass, quartz, or micaare usually used as hydrophilic substrates for microscopy applications. Transparent quartzsubstrates may be required when samples are analyzed using ultraviolet light.

Mica is a flat substrate at the molecular scale, useful for AFM applications. Mica sheetscan be easily cut to the desired size, and efficiently cleaned by just exfoliating the surface layerswith adhesive tape (although formation of micrometer-sized terraces could affect the obser-vation of the supported films at the optical microscopy scale). Microscopy cover slips are


good substrates for films to be analyzed by epifluorescence microscopy. New cover slips canbe washed with Chl/MetOH 3:1 (v/v) and dried in air to be clean enough. There is a wholevariety of alternative methods to clean recycled substrates. Chromic acid or saturated solu-tions of KaOH in water or ethanol, combined with ultrasonic treatment and water wash, maybe used for quartz or glass substrates. Rinse with a volatile solvent, such as isopropanol; later,N2 flow facilitates a rapid and homogeneous dry off.

3.2.2. Transfer to Solid Supports

The transfer of lipid monolayers is a suitable technique for the observation of lipid lateralsegregation in membrane-mimicking interfacial films. The major advantage of working withLB films is that the samples are immobilized and can be easily transported. This extends thepossibilities for their study by a combination of techniques not specifically designed forstudying monolayers in situ, including most spectroscopical techniques. One of the practicaldisadvantages of LB films is the obligation of preparing a different independent film to studythe structure at each of the surface pressures of interest. The target pressures must be selectedtaking into account the information from isotherm curves.

Pressures producing kinks and plateaus in the isotherms are good candidates to be relatedto lateral segregation processes. To identify pressures producing lipid segregation, the trans-fer of the monolayer may be performed while the monolayer is compressed. A precise con-trol of compression and transfer velocities allows obtaining LB films that contain all thestructures occurring along the isotherm on a unique support, with pressure being a functionof the position through all the transfer length.

A general protocol to transfer lipid monolayers to supports could be:

1. Clean carefully the surface balance.2. Add pure water or buffer as subphase.3. Clean the surface as indicated in Subheading 3.1.3.1. and the supports as suggested in

Subheading 3.2.1.4. With the barrier open at the largest area, mount the transfer plate on the lift system. Check that

the support is completely out of the bulk phase in the upper position, and that the full transfer areais immersed into the subphase at the lower position. Then, move the lift to the lowest position.

5. Close the barrier and clean the surface by aspirating the interface with a pipet connected to avacuum pump. If the substrate is perfectly clean, no changes must be observed on surface pressureduring compression.


Fig. 2. Different modes of preparing supported phospholipid films on transfer of interfacial mono-layers onto solid supports of different nature.

6. Spread the solution as in Subheading 3.1.3.1. and wait until solvent is totally evaporated.7. Select a proper compression speed and compress the monolayer to the desired target pressure.

To maintain the surface pressure constant along the transfer process, most of the commercialssurface balances include a feedback mechanism that automatically compensates with propercompression changes in the pressure owing to lipid extraction. The sensitivity and time-constantresponse of the feedback program must be carefully adjusted to avoid artefacts that can beobserved in the microscopy images as scratches in the film, usually orientated in a perpendicularway to the transfer direction.

8. Once the target pressure is reached, wait 10 min to allow for re-equilibration of the film. Whenlipid domains have been generated on compression, their shape usually changes from dendritic-like shapes just at the end of compression to characteristic equilibrium shapes at longer times(Fig. 3). Ten minutes is usually long enough to reach local equilibrium shapes in most systems.Liquid domains formed by lateral immiscibility (such as those formed by lipid mixtures con-taining cholesterol even at low pressures) are fluid enough to change substantially over time,often including domain coalescence (11). Therefore, the equilibration time is a very importantparameter when examining and comparing the morphology of films made of different mixturesor systems of this type.

9. While the feedback program is activated, raise the support until the lower edge is out of the bulkphase. A proper transfer speed is a crucial parameter to obtain well-transferred films without arte-facts. For each experiment, the optimal speed depends on the nature of the material forming themonolayer and the support dimensions. Transfer speeds of 5 mm/min are commonly used to obtainLB films from lipid monolayers. The progress of film transfer can be followed by plotting the reduc-tion in surface area that is required to maintain surface pressure constant on transfer against time.

10. Dismount the substrate from the lift and store the supported film at room temperature. To avoidlipid oxidation, storage of the LB’s in an inert atmosphere is recommended. When light-sensibledyes are included into the films, protect the LB’s in a dark environment.

11. To check for transfer efficiency, the deposition ratio may be calculated. This parameter is the ratebetween the final area reduction introduced at the interfacial monolayer to maintain constantpressure during transfer, and the area of the immersed support that has been lifted off the bulkphase. Films transferred with deposition ratios smaller than 0.9 must be discarded.

It is possible to stack more than one monolayer transferred into the same support, byrepeating the process after the first deposition (see Note 9). To facilitate the second transfer,


Fig. 3. Effect of equilibration on the shapes and morphology of condensed lipid domains. Epifluore-scence microscopy images obtained just after finishing compression of a DPPC film up to 12 mN/m (leftpicture) and after a 10 min equilibration of the film while maintaining the same pressure (right picture).Notice the higher branching level and the blurry boundaries of the domains before equilibration.

it is recommendable to let first the previous layer to dry out completely, in order to avoid thelipid of this first layer to be transferred again from the support back to the air–liquid inter-face. The structural information obtained from these multilayers is more difficult to interpretowing to overlapping in the structures of the different layers (see Note 10). To facilitate trans-fer of interfacial films onto hydrophobic substrates, the Langmuir–Schaefer method can beused (see Fig. 3). In this technique, the support is placed in contact with the monolayer thatis compressed at the desired pressure, and the film is removed horizontally from the interfaceat the appropriate speed.

3.3. Observation of Film Lateral Structures: Fluorescence Microscopy

Different techniques are available to analyze the structure of phospholipid-based interfa-cial films. Some of the spectroscopic techniques, such as grazing incidence X-ray diffraction,neutron scattering, or infrared reflection-absorption spectroscopy (9,12,13), report informa-tion that is averaged over the entire structure of the film. To obtain information on howlateral transitions take place or how the different lipid and protein components are laterallydistributed, some optical techniques are available providing enough spatial resolution to pro-duce images of the films. In this respect, fluorescence microscopy is one of the most usedtechniques (14–17). This technique needs for the inclusion of traces of fluorescent dyes intothe monolayers. Lateral inhomogeneities of the film structure are then revealed as far as theyare associated with differential probe solubility. The inclusion of these fluorescent dyes maybe considered a potential source of artefacts but the high sensitivity of the technique allowsobtaining good results with very-low dye concentrations. Some other techniques such asBrewster angle microscopy (BAM), ellipsometry, or scanning force microscopy have no needfor the use of dyes.

BAM takes advantage of the differences in the reflective properties of the interface and thedifferent regions of the films to obtain images of the interfacial structures, without requiringthe inclusion of exogenous probes (18). However, BAM has a relatively low lateral spatialresolution. Ellipsometry is a more sophisticated technique producing images of films fromthe changes in polarization introduced on reflection of light by the interfacial surfaces. Apartfrom detecting segregated structures, ellipsometry may provide information about somephysicochemical properties of the monolayers such as thickness, roughness, and so on (19).Fluorescence microscopy has some advantages when compared with these other techniques,the most important being that it has very high sensitivity, plus the possibilities derived fromthe inclusion of multiple dyes, which allows obtaining simultaneous information from thedistribution of different lipid and protein species.

3.3.1. Fluorescent Labels for the Observation of Lipid Segregation

Most dyes used for fluorescence microscopy of monolayer films are derivatives of mem-brane components, such as phospholipids, sphingolipids and cholesterol, or amphipathicmolecules structurally similar to any of these membrane components (20). In general, anymembrane-partitioning dye could be used to label interfacial monolayers, but molecules thatpartition differentially into different phases are the most useful when observation of domaindistribution is pursued. This differential partition of a given probe depends not only on itsstructure and intrinsic properties, but also on the potential interaction of the dye with the dif-ferent components forming the monolayer and on the physical state of the different regionsor phases existing in the film.


Bodipy, NBD, Fluoresceine (FL), dansyl, or Texas Red© (TR) (available from MolecularProbes, Junction City, OR) (see Note 11) are dyes frequently used to synthesize covalentlylabeled fluorescent phospholipid molecules (Fig. 4). The distribution of these modified lipidsin monolayers is highly dependent on where the dye is chemically attached at the phospho-lipid molecule. In principle, phospholipids bearing long saturated acyl chains and modifiedat the head group, can still be highly packed, remaining in LC or ordered phases at high pres-sures. In contrast, labels modified at the acyl chains of the molecule are frequently excludedfrom ordered phases. For instance, NBD-PC modified at one of the acyl chains of the molecule,is typically excluded from ordered phases of DPPC monolayers (8,16). In contrast, a phos-pholipid modified at the headgroup such as NBD-dipalmitoylphosphatidylethanolamine(NBD-DPPE), can still remain in packed LC domains of DPPC monolayers.

However, the behavior of different probes cannot be easily predicted based only on theirstructural location. Probes such as TR-DPPE or FL-DPPE, which are also labeled at thehead group, are effectively excluded from LC regions owing to the bulky structure of the flu-orescent group (21). In general, the distribution of the different dyes must be understoodtaking into account the properties of the different lipid phases analyzed and the “in-planesolubility” of the probe within each phase. As stated in Subheading 2.1., the use of label


Fig. 4. Structure and typical orientation of different fluorescent phospholipid derivatives at theair–liquid interface.

concentrations more than 1% (M) is not recommended when avoiding effects on the mono-layer structure is pursued (see Note 12).

Other available amphiphilic molecules such as dialkycarbocyanines, Laurdan, or Prodan(i.e., from Molecular Probes, Junction City, OR) may also be used. The octadecyl indocarbo-cyanine DiIC18 is frequently used to label ordered phases. The emission spectra of prodan andlaurdan depends on the orientation of the dye in the membranes, making it possible to obtainmeasurements of fluorescence anisotropy from defined regions of the layers (22,23).

Some other probes could be used in more specific applications. Specific interactions ofproteins with monolayers have been analyzed using fluorescent derivatives of certain pro-teins. Combination of ganglioside GM1 and fluorescein-conjugated cholera toxin B havebeen used to detect raft-like domains in monolayers and bilayers (21,24,25).

Combination of two or more probes could be used to label different domains simultane-ously (26,27). In these experiments, the excitation and emission spectral properties of the dif-ferent probes must be taken into consideration to avoid or to take advantage of fluorescentresonance energy transfer processes occurring in the films.

3.3.2. Capturing Images for Analysis

To obtain images from fluorescently labeled interfacial films, two possibilities can beconsidered: either a direct observation of the monolayer in situ at the air–liquid interface orobservation of immobilized transferred monolayers.

3.3.2.1. MONOLAYERS “IN SITU”

The films can be directly observed at the interface in situ, directly focusing at the interfaceusing a fluorescence microscope installed over the surface balance. The trough in this set-up mustbe well isolated from vibrations to avoid changes in the focus plane during image capture. Theexperiments have to be conducted in dark environments to protect fluorescent dyes from lightbleaching. In some troughs, an inverted microscope could be used to watch the interface througha glass window installed at the base of the container, and located only at a few millimetres fromthe air–liquid interface to allow proper focusing. It is important to take into account that even inthe absence of stirring, different phenomena including convection, evaporation, and bulk phasedisplacements initiated by the mechanical movements of the barrier, make the liquid surface tobe in permanent motion. As a consequence of this, the interfacial film is usually experimentingfast lateral diffusion, except when the interface is occupied by highly packed material.

Most of the photographic cameras use exposure times that are too long to obtain goodfrozen images from the fluorescence of a monomolecular monolayer under continuous move-ment. Observation of films in situ therefore critically requires that a highly sensitive intensi-fied CCD camera (i.e., from Andor Technology, South Windsor, CT, or Hamamatsu PhotonicsK.K., Hamamatsu, Japan) be available to record short videos from the interface that can belater used to extract independent frames. Simultaneous observation of different fluorescentprobes in the same frames of those fast-moving films is a technical challenge. To minimizemovements, the small region of the interface just under the microscope can be confined intoa small compartment connected to the rest of the interface through a narrow channel; caremust be taken to ensure that the connecting channel is allowing for equilibration of the mostviscous films, such as phospholipid monolayers compressed to the highest pressures.

When image capturing is performed in situ, the changes in the structure of the film andthe lateral distribution of fluorescence can be recorded from an entire Π-A or Π-timeisotherm, using a unique film compressed to any desired pressure. Once compression has


been stopped at the target pressure, some time of equilibration (2–5 min) is usually waitedbefore starting video capturing.

3.3.2.2. TRANSFERRED MONOLAYERS

Once an interfacial film compressed to a given pressure is transferred to a solid support,the resulting LB film needs less technical requirements to be observed under fluorescencemicroscopy. These samples are completely immobilized allowing easy observation of over-lapping regions using different filters, which facilitates the use of dye combinations in thefilms to localize specific regions or exploring for colocalization of different components.Transferred monolayers may also be subjected to different treatments after the transfer, suchas blotting or staining. For instance, treatment with detergents of LB films made fromdioleoylphosphatidylcholine (DOPC)/cholesterol/sphingomyelin mixtures has been used todetect formation of detergent-resistant “raft-like” domains (21).

The main disadvantage of observing support-immobilized films is their rapid fluorescencephotobleaching under the microscope as a consequence of sample immobilization. Therefore,it is important to limit the time of exposure during image collection. It may be enough toobtain a few images per frame of film, yet the observed region gets irreversibly burned andremains unusable for further observations.

It is also important to take into account that the LB method described in Subheading 3.2.2.produces films transferred at both sides of the substrate. Focusing on just one of the mono-layers could be a problem. To ensure the observation of the right surface, it is recommendedto carry out the transfer using two supports coupled side by side. Once the transfer has beenfinished, one of the two supports (preferably the one immobilizing the film that was behindthe scan direction of the barrier) should be discarded.

3.3.3. Analysis of Lipid Segregation in Interfacial Films

3.3.3.1. QUALITATIVE EVALUATION OF LATERAL FILM REORGANIZATION

Information acquired from the isotherms of phospholipid films compressed to differentpressures may be used to build phase diagrams. In the simplest case, Π A isotherms obtainedat different temperatures may give enough information to construct Π vs temperature phasediagrams. Transition pressures obtained from horizontal plateaus or kinks observed in theslope of the isotherms are used to determine boundaries between the phases in the diagram.

Complementary information obtained from epifluorescence microscopy is useful to com-plete the structural information reported by the phase diagram. Observation of the apparentmorphology of segregated lipid domains may allow, for instance, distinguishing between aLC/LE coexistence and one of liquid-ordered-like (LO)/LE type (see Fig. 5).

Phase diagrams, as isotherms, are very dependent on the structural properties of the phos-pholipid. The temperature and the surface pressure at which the different lateral transitionsoccur are parameters very much dependent on the length and/or the saturation state of thephospholipid acyl chains, in a similar way as they affect the fluid–gel phase transitions inmembranes. Phospholipids with longer acyl chains have transitions at higher temperaturesand lower pressures than those with shorter acyl chains. Increasing the length of the chainby a single methylene group is enough to increase by 5–10°C the temperature of its transitionand decrease by about 10 mN/m the surface pressure (9,28). Phase diagrams may also be


affected by subphase properties, such as pH or presence of ions, which could affect interac-tions between charged lipid head groups in the monolayers.

To analyze lateral lipid segregation in monolayers, representation of composition-pressurephase diagrams is also useful. In this case, structure-sensitive techniques such as epifluorescenceare the most useful because immiscibility is not easily detected exclusively from the informa-tion reported by isotherms (see Note 13). In binary systems, typical phase diagrams of mis-cibility pressures vs the mole fraction of one of the components are used (see Note 14). Whensystems containing three components are analyzed, ternary diagrams can be obtained frombinary diagrams of two of the components at different constant molar fractions of the thirdone. As an example, liquid–liquid phase coexistence occurring in binary and ternary mixturesof dihydrocholesterol and phospholipids have been studied from their composition-pressurephase diagrams, as models to understand the lateral segregation thought to occur in real cellmembranes (29,30).

3.3.3.2. QUANTITATION OF COMPRESSION-DRIVEN LIPID-SEGREGATION

Additional quantitative information may be obtained from epifluorescence images toanalyze the effect of different factors on the phase transition, along the compression isotherm.Quantitative analysis of lipid segregation can be made by computer-assisted estimation of theamount of fluorescent/nonfluorescent areas, with respect to the particular lateral distributionof any probe. Different image-analyzing programs are available to process digitally capturedimages and obtain quantitative data that include statistics of domain sizes or perimeters, thetotal fraction of condensed area, and the density of domains. NIH Image and ImageJ are freeprograms from the National Institute of Health for the Macintosh or Java platforms, respec-tively (see Note 15). A PC version of this program, Scion Image, is also available from ScionCorporation (MD). Data analysis requires processing of at least 10 frames per surface pressureto obtain statistical results.


Fig. 5. Different examples of lipid phase and domain segregation as observed by epifluorescencemicroscopy of interfacial films. (Left panel) Typical round-shaped domains in PC/sphyngomyelin/Cholmixtures (24:45:30 w/w/w) exhibiting fluid/fluid immiscibility. (Central panel) chirality-shaped,LC, ordered domains of DPPC films compressed into the LE/LC plateau. (Right panel) DPPC andChl-enriched liquid-ordered-like condensed domains segregated in pulmonary surfactant films com-pressed more than 30 mN/m.

Typical data reported from the quantitative analysis of images are:

1. Average percent of condensed area vs surface pressure.2. Domain area vs surface pressure.3. Number of domains per frame vs surface pressure.

Any component interacting with the phospholipid monolayer can affect one or several ofthese curves depending on the character of the interaction. Some components such as certainproteins or peptides may interact with the monolayer without altering the mean proportion ofcondensed phase observed along the transition. However, there is a reduction in size andincrease in number of the condensed domains. This type of effect is usually interpreted as a con-sequence of the interaction of the added component with condensed domain boundaries, whichproduces a reduction in the line tension (see Note 16). An alternative explanation is that theadditive may act as a center for nucleation of phospholipid condensates during compression.Molecules such as peptides or proteins interacting deep into the acyl chain region of phospho-lipid layers do usually affect both the domain distribution and the proportion of total condensedphase at any surface pressure along the compression isotherm. The interpretation of this effectis similar to that of proteins removing enthalpy from the gel-to-fluid thermotropic transition inlipid–protein suspensions studied by calorimetry. Interaction of proteins with the acyl chains ofphospholipids subtracts lipid molecules from undergoing compression-driven condensation, ina proportion defined by the lipid/protein stoichiometry.

Care must be taken when quantitative data on lipid segregation are used to discuss membrane-relevant features because size, shape, and number of condensed domains are very dependent onthe way the monolayer is prepared and compressed. As an example of this, the faster the mono-layer is compressed, the smaller is the size of the lipid domains. Another consequence of thisfeature is that in monolayers prepared from adsorbed material, the size of the domains is alsodependent on how rapid the material adsorbs into the air–liquid interface.

4. Notes1. Alternatively, some balances permit a more isotropic compression by incorporating two PTFE

barriers that reduce the surface available to the film in a more homogeneous way around the surfacetension measurement point. This approach may be particularly useful when observing simultane-ously the distribution of fluorescent probes at the surface, because it may largely reduce the lateralmovements owing to displacement by the barrier of the upper layers of the subphase.

2. To maintain the subphase volume and avoid changes because of evaporation, a feedback systemconsisting of a buffer reservoir connected to the bulk phase, and an ellipsometric device detectingchanges on the surface level may be used.

3. Surface pressure (Π) is defined as the reduction on surface tension of the clean liquid phase pro-duced by the monolayer at the interface.

4. The Wilhelmy method uses a plate or flag partially immersed in the subphase and suspended froma balance to measure surface tension. The force measured depends on the weight of the plate, thebuoyant force, and is a function of the surface tension (γ). The force produced by surface tensionover the flag depends on the contact perimeter and on the cosine of the contact angle of theWilhelmy plate with the interface. The contact perimeter can be easily determined; the contactangle is negligible when the flag is wet enough. To improve wetting of platinum Wilhelmy flags,these must be well scratched and completely immersed in water before raised to its final position.This step is not necessary for paper plates if they were previously immersed in water.

5. The compression rate of the film in Π-A isotherms should be always given as normalized areaper molecule per time unit, for the isotherms to be directly comparable with those obtained indifferent troughs.


6. A very slow compression rate (in the order of 2 Å/mole/min) allows obtaining the isothermsunder quasi-static equilibrium conditions. In thermodynamical terms, such isotherms are muchbetter interpreted than isotherms recorded under dynamic conditions, but they may reflect poorlysome of the phenomena occurring in living membranes. It is important to check how dependentis the structure of lipid films at defined pressures (i.e., at the membrane relevant pressure of~30 mN/m) on the compression rate used to reach them.

7. The area per molecule at this “lift-off” pressure of the isotherm is a characteristic structuralparameter, as it can be related to the maximal surface occupied by a single molecule (its molec-ular shape) in the absence of compression.

8. Ordered phases such as LC or the solid phase are typically reached by films made of phospho-lipids bearing saturated acyl chains at temperatures lower than their gel-to-fluid melting Tm. Iftemperature is higher than Tm, the lipid is intrinsically too disordered to be condensed and thefilms collapse when certain pressure is reached directly from the LE phase (see Fig. 1).

9. A second transferred lipid film could allow preparation, for instance, of supported bilayers,wherein the lateral distribution of lipids and proteins could be analyzed in a disposition thatmimics free-standing membranes better than that of pure monolayer LB films. In these sup-ported bilayers, the lipid and protein molecules taking part of the second layer diffuse in theplane of the film more freely than those directly in contact with the support.

10. When forming multilayer arrays, the orientation of the first layer determines the subsequenttransferences. In “Y-type” arrays, which are the most common including those mimicking bio-logical membranes, any layer has an opposite orientation to the surrounding ones. Less fre-quently, all the layers can be oriented in the same direction. These films are called X- or Z-type,depending on whether the outermost side is hydrophilic or hydrophobic, respectively.

11. Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), NBD, fluorescein (spiro[isobenzofuran-1(3H),9′-(9H)-xanthen]-3-one, 3′,6′-dihydroxy), dansyl (5-dimethylaminonaphthalene-1-sulfonyl),Texas Red (1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium,9-[2(or4)-(chloro-sulfonyl)-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro).

12. Recent experiments using high-resolution techniques such as AFM to compare the structureof probe-free and probe-containing phospholipid films at both micro- and nanoscopic scales,have allowed detection of effects produced by the dye on monolayer nanostructure even atlow concentrations (0.5–1% M) (14). This suggests that the proportion of exogenous probesshould be maintained as low as possible, and their effect evaluated extensively by comple-mentary techniques.

13. It is important to consider that detection of lipid segregation in compressed interfacialmonolayers is only possible at the level of resolution provided by the particular microscopytechnique used. Observation of fluorescent probes by epifluorescence microscopy in situallows detection of lipid domains not much smaller than 1 µm. The actual size of the differenttypes of membrane domains reported in the literature is a matter of discussion, but differentauthors argue that domains segregated in lipid mixtures mimicking the real complexity ofmembranes may have sizes ranging more in the nanometer than in the micrometer scale.Fluorescence observation of immobilized LB films improves resolution a bit, but AFM offilms transferred onto mica or silicon is a much better technique to observe domains of onlya few nanometers in diameter.

14. Miscibility pressure is the maximal surface pressure at which ordered domains are still segregated.15. NIH Image and ImageJ are available for downloading at: http://rsb.info.nih.gov/. Scion Image

may be downloaded from Scion Corporation home page at http://www.scioncorp.com/.16. Line tension is the net component of forces experimented by the molecules at the boundaries of

2D segregated domains in interfacial films. Size and shape of segregated domains have beeninterpreted as a function of the dipolar interactions between the molecules taking part of thedomains, and the line tension at their boundaries. Line tension is what makes fluid domains toadopt a circular shape in the films, as surface tension minimizes the exposed surface of soapmicelles or oil drops in 3D emulsions.


AcknowledgmentsResearch in the laboratory of the authors is founded by grants from the Spanish Ministry

of Science and Education (BIO2006-03130) and Community of Madrid (S-0505/MAT/0283).The research group at Universidad Complutense also participates in Marie Curie NetworksEST-007931 and RTN-512229.

References1. Harder, T. (2003) Formation of functional cell membrane domains: the interplay of lipid- and

protein-mediated interactions. Philos. Trans. R. Soc. London B 358, 863–868.2. Helms, J. B. and Zurzolo, C. (2004) Lipids as targeting signals: lipid rafts and intracellular traf-

ficking. Traffic 5, 247–254.3. Simons, K. and Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol.

1, 31–39.4. Taneva, S., McEachren, T., Stewart, J., and Keough, K. M. (1995) Pulmonary surfactant protein

SP-A with phospholipids in spread monolayers at the air-water interface. Biochemistry 34,10,279–10,289.

5. Nag, K., Perez-Gil, J., Cruz, A., Rich, N. H., and Keough, K. M. (1996) Spontaneous formation ofinterfacial lipid-protein monolayers during adsorption from vesicles. Biophys. J. 71, 1356–1363.

6. Launois-Surpas, M. A., Ivanova, T., Panaiotov, I., Proust, J. E., Puisieux, F., and Georgiev, G.(1992) Behavior of pure and mixed DPPC liposomes spread or adsorbed at the air-water inter-face. Colloid Polym. Sci. 270, 901–911.

7. Cruz, A., Worthman, L. A., Serrano, A. G., Casals, C., Keough, K. M., and Perez-Gil, J. (2000)Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphos-phatidylcholine interfacial films spread from lipid-protein bilayers. Eur. Biophys. J. 29, 204–213.

8. Nag, K., Boland, C., Rich, N., and Keough, K. M. (1991) Epifluorescence microscopic observa-tion of monolayers of dipalmitoylphosphatidylcholine: dependence of domain size on compres-sion rates. Biochim. Biophys. Acta 1068, 157–160.

9. Kaganer, V. M., Möhwald, H., and Dutta, P. (1999) Structure and phase transitions in Langmuirmonolayers. Rev. Mod. Phys. 71, 779–819.

10. Marget-Dana, R. (1999) The monolayer technique: a potent tool for studying the interfacialproperties of antimicrobial and membrane-lytic peptides and their interactions with lipid mem-branes. Biochim. Biophys. Acta 1462, 109–140.

11. Samsonov, A. V., Mihalyov, I., and Cohen, F. S. (2001) Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes. Biophys. J. 81, 1486–1500.

12. Peng, J. B., Barnes, G. T., and Gentle, I. R. (2001) The structures of Langmuir-Blodgett films offatty acids and their salts. Adv. Colloid Interface Sci. 91, 163–219.

13. Wang, L., Cai, P., Galla, H. J., He, H., Flach, C. R., and Mendelsohn, R. (2005) Monolayer-multilayertransitions in a lung surfactant model: IR reflection-absorption spectroscopy and atomic forcemicroscopy. Eur. Biophys. J. 34, 243–254.

14. Cruz, A., Vazquez, L., Velez, M., and Perez-Gil, J. (2005) Influence of a fluorescent probe on thenanostructure of phospholipid membranes: dipalmitoylphosphatidylcholine interfacial monolayers.Langmuir 21, 5349–5355.

15. Nag, K., Perez-Gil, J., Ruano, M. L., et al. (1998) Phase transitions in films of lung surfactant atthe air-water interface. Biophys. J. 74, 2983–2995.

16. Perez-Gil, J., Nag, K., Taneva, S., and Keough, K. M. (1992) Pulmonary surfactant protein SP-Ccauses packing rearrangements of dipalmitoylphosphatidylcholine in spread monolayers.Biophys. J. 63, 197–204.

17. von Tscharner, V. and McConnell, H. M. (1981) An alternative view of phospholipid phase behaviorat the air-water interface. Microscope and film balance studies. Biophys. J. 36, 409–419.


18. Kaercher, T., Honig, D., and Mobius, D. (1993) Brewster angle microscopy. A new method ofvisualizing the spreading of Meibomian lipids. Int. Ophthalmol. 17, 341–348.

19. Bakowsky, U., Rothe, U., Antonopoulos, E., Martini, T., Henkel, L., and Freisleben, H. J. (2000)Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at thewater-air interface. Chem. Phys. Lipids 105, 31–42.

20. Maier, O., Oberle, V., and Hoekstra, D. (2002) Fluorescent lipid probes: some properties andapplications (a review). Chem. Phys. Lipids 116, 3–18.

21. Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., et al. (2001) Lipid rafts reconstituted in modelmembranes. Biophys. J. 80, 1417–1428.

22. Bagatolli, L. A. and Gratton, E. (2000) Two photon fluorescence microscopy of coexisting lipiddomains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys. J. 78, 290–305.

23. Krasnowska, E. K., Bagatolli, L. A., Gratton, E., and Parasassi, T. (2001) Surface properties ofcholesterol-containing membranes detected by Prodan fluorescence. Biochim. Biophys. Acta1511, 330–340.

24. Bacia, K., Scherfeld, D., Kahya, N., and Schwille, P. (2004) Fluorescence correlation spec-troscopy relates rafts in model and native membranes. Biophys. J. 87, 1034–1043.

25. de Almeida, R. F., Loura, L. M., Fedorov, A., and Prieto, M. (2005) Lipid rafts have differentsizes depending on membrane composition: a time-resolved fluorescence resonance energytransfer study. J. Mol. Biol. 346, 1109–1120.

26. Nag, K., Taneva, S. G., Perez-Gil, J., Cruz, A., and Keough, K. M. (1997) Combinations of flu-orescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films. Biophys. J.72, 2638–2650.

27. Ruano, M. L., Nag, K., Worthman, L. A., Casals, C., Perez-Gil, J., and Keough, K. M. (1998)Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers ofdipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidyl-glycerol. Biophys. J. 74, 1101–1109.

28. Mohwald, H. (1990) Phospholipid and phospholipid-protein monolayers at the air/water inter-face. Annu. Rev. Phys. Chem. 41, 441–476.

29. Keller, S. L., Anderson, T. G., and McConnell, H. M. (2000) Miscibility critical pressures inmonolayers of ternary lipid mixtures. Biophys. J. 79, 2033–2042.

30. McConnell, H. M. and Radhakrishnan, A. (2003) Condensed complexes of cholesterol and phos-pholipids. Biochim. Biophys. Acta 1610, 159–173.


Date post:	23-Dec-2016
Category:	Documents
Upload:	alex-m
View:	213 times
Download:	0 times

[Methods in Molecular Biology™] Methods in Membrane Lipids Volume 400 || Langmuir Films to...

Documents